Ca2+, diacylglycerol and phosphoinositide second messengers are among the most versatile mediators that link many if not most human receptor inputs to diverse cell function. Several of the associated signaling steps are known to be impaired in a wide range of immunological, heart and brain diseases. What is poorly understood is how Ca2+, diacylglycerol and PI (4, 5) P2 signals work synergistically with the recently discovered ER-PM junctions and with cortical actin to regulate fundamental multi-step cellular processes such as secretion, cell migration and proliferation. Past work in the field focused on identifying molecular mechanisms that generate Ca2+, diacylglycerol and PI(4,5)P2 signals including our discovery of the ER-Ca2+ sensors STIM1 and STIM2 that control plasma membrane Ca2+ influx. With the core mechanisms now being largely understood, a huge challenge has emerged to determine which of the many targets regulated by Ca2+, diacylglycerol or PI (4, 5) P2 signals are important and how the relevant targets dynamically orchestrate specific cellular processes. To overcome this challenge, our laboratory has developed a unique set of tools to monitor and manipulate Ca2+, diacylglycerol and PI (4, 5) P2 along with the interconnected ER-PM junctions and cortical actin. In addition, we have established during the last funding period single cell model systems and assays tailored for systematic investigations of the common and specific roles of these signals in secretion, cell migration and the cell cycle. With these methods in place and by building on some of our initial results, our proposed work will establish how ER-PM junctions are formed and what roles they have, particularly in the context of secretion, migration and proliferation. Furthermore, our study will lead to systems level insights into universal and specific roles of the Ca2+, diacylglycerol and PI (4, 5) P2 control system in driving secretion, migration and proliferation. Finally, our wor will also provide the Ca2+ research community with broadly useful research tools.